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3D CELL CULTURE TECHNOLOGY www.celvivo.com
With ClinoStar®, we built a cutting-edge
system that provides scientists with superior
growth conditions, to implement spheroid
and organoid culture in their labs.
2
2
21 day old spheroids of HEPG2/C3A cells
cultivated in ClinoStar®
3D CELL CULTURE
INCREASE THE
PERFORMANCE
OF YOUR CELL LINE
ClinoStar® was built because we needed a
better model for our research. The success
criteria was a system which was easy to
operate and could create abundant and
reproducible models with
in vivo functionality.
ClinoStar® creates an environment that
promotes longevity and creates uniform
cell constructs
The ClinoStar® system is a 3D bioreactor platform that creates an environment,
which promotes growth, maintenance and functionality of large 3D tissue
mimetic structures, including spheroids, organoids and other cell aggregates.
It enables conditions, which allow cells to develop functionality that closely
mimics conditions in the intact organism.
3
CLINOSTAT FOR ClinoReactor ClinoStar®
CELL CULTURE
The system employs a clinostat and a rotating bioreactor
to keep the cells suspended by counterbalancing the
gravitational forces.
Basic Principle
This approach has the advantage that the spheroids and
organoids formed, are exposed to very low shear forces FORCE
BY UPLIFT
and have good gas and nutrient exchange. Low shear MOVEMENT
forces, combined with active diffusion, are key parameters 1
2
for cells to develop into functional 3D constructs.
3 B
CLINOSTAT PRINCIPLE ACTIVE DIFFUSION 1) cells; 2) culture media; 3) culture vessel
Basic Principle
Basic Principle
Passive diffusion
FORCE 175 µM radius spheroid
FORCE
BY UPLIFT BY UPLIFT
MOVEMENT MOVEMENT MEDIA FLOW
1 1 Nutrients
2 2 Nutrien
Di
ffu
sion
Metabolic
depleted
Metabolic
waste waste
FORCE FORCE
3 BY GRAVITY 3 BY GRAVITY
zo
ne
02 part
pressu
02 partial
1) cells; 2) culture media; 3) culture vessel pressure MEDIA FLOW
1) cells; 2) culture media; 3) culture vessel
Basic Principle
Passive diffusion Active diffusion
175 µM radius spheroid Passive
450 µMdiffusion
radius spheroid Active diffusion
175 µM radius spheroid 450 µM radius spheroid
FORCE
LOW SHEAR FORCESMEDIA FLOW BY UPLIFT
Nutrients MOVEMENT MEDIA FLOW
NutrientsNutrients
1
2 Nutrients
Di
ffu
Di
sion
ffu
sion
Metabolic
depleted
Metabolic
waste Metabolic waste
depleted
Metabolic
waste waste
FORCE
zo
3 BY GRAVITY
ne
zo
02 partial
ne
pressure
02 partial 02 partial
pressure MEDIA FLOW pressure
02 partial
pressure MEDIA FLOW
1) cells; 2) culture media; 3) culture vessel
Passive diffusion With the clinostat principle, the spheroids
Active or
diffusion
175 µM radius spheroid 450 µM radius spheroid
organoids will experience continued media flow,
The gentle rotation (2.5-30 rpm) created by the abolishing theFLOW
MEDIA diffusion depleted zone observed
Nutrients
clinostat, ensures the spheroids or organoids with static cultures. This active diffusion across the
Nutrients
are kept in statical orbit with low shear stress cell conglomerates, allows for better nutrient and
Di
ffu
introduced. In this stress-free environment, the gas exchange, essentially allowing the spheroids
sion
cells have time to self-organize and maintain or
Metabolic and organoids toMetabolic
develop to the necessary size
depleted
waste
re-establish their architecture and functionality to and to mimic nativewaste
cytoarchitecture and display
resemble their parental tissue. physiological attributes of the native tissue.
zo
ne
02 partial
pressure
02 partial
4 pressure MEDIA FLOW
FEATURED
FRIENDS
Our featured friends are ”We are adapting all of our projects
a group of experienced using spheroids. They model more
accurately every aspect of cell
scientists that have used physiology compared to canonical flat
the ClinoStar® prototype cultures, and this has allowed us to
in their own research. eliminate part of animal testing from
See what they think or our workflow. We are really impressed
by how simple it is to maintain and
read their publications.
prevent contaminations of these
spheroids for both experienced and
inexperienced users.”
Simone Sidoli - Assistant Professor
Department of Biochemistry at the
Albert Einstein College of Medicine
To find out more about Simone
and his research, scan the QR Code
”Spheroids created with CelVivo ”Using clinostat-based 3D cultures
system have become an enables much longer treatment
indispensable tool in my research and experimental windows, in an in
combining proteomics and lipidomic vitro format, while obtaining data
to understand the role of protein with physiological relevance. Unlike
oxidation and redox imbalance in cell with animal models, multiple daily
physiology induced by drugs samplings from the same bioreactor is
and xenobiotics.” possible, with no ethical implications.
I believe this is an ideal approach.”
AdelinaRogowska-Wrzesinska-AssociateProfessor
Department of Biochemistry and Molecular Biology,
Chrisna Grouws - Associate Professor
University of Southern Denmark
Pharmaceutical and Biomedical Pharmaceutics,
To find out more about Adelina North-West University
and her research, scan the QR Code
To find out more about Chrisna
and her research, scan the QR Code
5
CLINOSTAR®
FEATURES &
BENEFITS
EASY CLEAN
The ClinoStar® is a premium class CO2 CORNERLESS
incubator with six independent motors DESIGN
(clinostats), which each can hold a
bioreactor (ClinoReactor). The system
is operated using a tablet* with pre-
installed software that permits control
of the temperature and CO2 level of each
ClinoStar® independently. Six camaras
located opposite to the motors enable
video surveillance of the cultures
without disturbing the environment.
Push to open
For convenient hands free opening
and reduced contamination risk.
Adjustable light
Front and backlight can be adjusted
to obtain crystal clear images.
Small footprint
Fits anywhere, even in your laf-bench.
Uniform environment
The large heating element and fan
ensure an equal distribution and
low variability of heat and CO2
across the chamber.
Connectivity options
An ethernet connection allows for
direct internet access or receive
software updates via the control unit.
Decontamination
Automatic UVC-decontamination
cycles.
*Supplied with the starter pack.
6
Remote Control
Control, adjust and monitor your cultures.
One Tablet can control up to 50 units.
Software over the air
New features and tools
are implemented by
software updates.
Live Camera Feed
No need to open to the
door. Track your culture
through the 5MP camera.
Enable 6 individual
x6 experiments
The six motors can be
individually adjusted.
DIMENSIONS
SEE PAGE 15
7
SEALED,
WRAPPED
CLINOREACTOR
AND READY
TO USE!
FEATURES &
BENEFITS
ClinoReactor is a bioreactor with a fixed 10 mL
culture chamber, that is supplied sterile in a sealed
and wrapped package for convenient use and to
limit the infection risk. The humidification beads
supply water for the culture chamber, which
eliminate the need for water in the incubator.
To prevent cell, protein and drug absorption,
the bioreactor is made from low bind polypropylene
and polystyrene. The optically clear properties of the
polystyrene enables microscopy of the spheroids
or organoids in a closed environment.
Fixed 10 mL cell culture chamber
Can contain and maintain over 350 mature
cell constructs.
Low bind surface
Polypropylene and polystyrene surfaces ensure a
low adhesion and absorption of molecules.
Click-on
Simple click-on system for easy placement and
removal of ClinoReactors in ClinoStar®.
Unique airflow and filter system
0.2 µm filter protect the cultures from contaminants
while gas exchange is maintained, enabling use of
bicarbonate buffered media.
Free standing
Integrated feet at the base of the ClinoReactor
provides a stable foundation when
exchanging media.
8
Petri dish accessibility
Scan the QR Code to see the
functionality of the petri dish opening.
Optically clear lid
Can be placed directly under the microscope.
Closed environment
Contained humidification system maintain a
constant volume in the chamber and limit the risk
of infections spreading.
Easy media exchange through the top port
PRODUCT Simultaneous media exchange for all cell
DATA
SEE PAGE 15 constructs, performed within minutes.
9
HOW TO WORK
WITH CLINOSTAR®
Culturing spheroids and organoids is dependent on the cell model and
sample being cultured. With the ClinoStar® system, we have created a
generic system to generate spheroids and organoids in a reproducible
fashion. The initial cell aggregation, can be performed in several ways
to accommodate various cell and sample types, but the downstream
process remains. Each step, breaks down the process of generating
reproducible spheroids and organoids with ClinoStar®.
Spheroid and organoid development
1. Select aggregation method
Following 2D cell expansion, the cells can be seeded in the
ClinoReactor with one of three approaches: single cell suspension
or initial aggregates preformed in hydrogel or micropattern plates.
2. Transfer to ClinoReactor
In the ClinoStar® system, you can use your regular cell culture
media and supplements, obviate the need for specialised media,
scaffolds, ECM substitutes or growth factors.
3. Cultivate in ClinoStar®
In the cultivation phase, cell culture media is routinely renewed,
and irregular constructs are removed from the ClinoReactor to
ensure uniform growth. Check that your constructs are functional
against a parameter that can be assayed in vivo.
4. Mature the constructs
When the cell lines have recovered their functionality, they are
considered mature and ready for experiments. For HEPG2/C3A cells
each ClinoReactor will hold around 350 spheroids after 18 days
of maturation.
10BUILT TO AVOID
CONTAMINATION
Infections are one of the main
challenges in cell culture and it
can cause significant delays.
A series of features in the ClinoStar® system have limited the cell
cultures contact with their surroundings, removed unnecessary
contamination points and made it easy to clean.
EASY REDUCED PROTECT
CLEANING EXPOSURE AND CONTAIN
Corner-less design Push to open Double wrapped
Easy cleaning though out No contact with hands Open directly into the
the experiment sterile workspace
Camera monitoring
UV decontamination Clear view without opening Easy clean ClinoReactor
Run regular The collar is easily disinfected
sterilisation cycles with ethanol
Solidified humidification
Seperate humidification
Glass and smooth surfaces system in each ClinoReactor Infection is contained
Disinfection and cleaning The closed design ensures
with ethanol infections are not spreading
110.5 0.5
0 0
0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 40 45
Days *mM/day/total cellular protein Days
CLINOSTAR® PROMOTES60 60
CELL FUNCTIONALITY
50 3.0
50 3.0
2.5 2.5
(µM)*
Cholesterol (µM)
40 40
in vivo level
in vivo level
Cholesterol
30 2.0
30 2.0
Urea (mM)*
(mM)
For a model to have the highest value, it must provide Both cholesterol and urea* are primarily synthesised in
20 1.5
20 1.5
the liver, while ATP is synthesised in every living cell. In
Urea
an accurate representation of human physiology in vivo.
10
all three
1.0
10
situations, we found that it took about 18 days 1.0
Many cells, when grown in 2D cultures have doubling for the rate of synthesis to increase to levels seen in vivo.
times 0 of 1-2 days. In contrast, cells in tissues for After0.50this time, the rates stabilised – at least for the next 0.5
0 10
example the liver, have a20doubling
30
time of40
200 – 30050 24 days0 (and probably 10 20
much 30 This provides
longer). 40 a 50
Days 0
*µM/day/total cellular protein Days 0
days (the rate needed to maintain the tissue). In 2D large window
0 5 for10 experimentation
15 20 25 where
30 spheroids
35 40 are
45 0
cultures, HepG2/C3A cells – a cell line derived from at a metabolic equilibrium, Figure Days 1. *mM/d
a hepatocellular carcinoma has a doubling time of
about 1 day (top left figure). If those cells are grown as For this reason – and for the sake of convenience, we
5.0 5.0
60 60
spheroids in a ClinoReactor, their rate of proliferation therefore recommend that – for the C3A cell line –
4.5 4.5
slows dramatically so that after 42 days in culture , their spheroids
50
are cultivated for 21 days if one wishes 50
4.0 4.0
doubling time is about 70 days (much closer to the to mimic in vivo physiology. Other cells (whether
in vivo level
vivo level
3.5 3.5
primary
40 or pluripotent cells or cell lines) may reach this
Cholesterol (µM)*
situation in vivo).
(µM)
40
3.0 3.0
level
ATP (mM)*
equilibrium at different times and so it is important to
ATP (mM)
Cholesterol
in vivo in
2.5 2.5
30 30
We investigated three physiological functions seen in benchmark against the in vivo performance
the2.0
liver and compared the performance of spheroids of 2.0
(Wrzesinski et al. 2013).
20 20
1.5 1.5
different ages with that of the same number of cells in
1.0
an adult human, Figure 1. 1.0
10 10
0.5 0.5
0.0 0
0.0 0
0 5 10 15 20 25 30 35 40 45 0
0 5 1010 and15urea 20 25 30 30
20 production 35 40 45
Figure 1. Charataristics of a HepG2/C3A cells cultivated in ClinoStar®. Doubling time, ATP content, cholesterol have been40
evaluated at50 0
multiple timepoints up to the endpoint at 42 days (Wrzesinski et al. 2013).
Days *mM/day/total cellular protein Days *µM/d
Days in culture
3.0 3.0
0 5 10 15 20 25 30 35 40 45
0 5.0 5.0
2.5 2.5
10 4.5 4.5
in vivo level
level
20 4.0 4.0
level
2.0 2.0
(days)
in vivo
(mM)*
30 3.5 3.5
time(mM)
in vivo
40
1.5 3.0
1.5 3.0
ATP (mM)*
(mM)
DoublingUrea
ATPUrea
50 2.5 2.5
1.0 1.0
60 2.0 2.0
70 1.5 1.5
0.5 0.5
80 1.0 1.0
0
90 0
0.5 0.5
0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 30 35 40 45
100 0.0 0.0
Days in vivo turnover 200-300 days *mM/day/total
0 5 cellular
10 protein
15 20Days25 30 35 40 45 0
Days *mM/d
60 3.0
60 3.0
Days in culture
50 50 0
2.5 5 10 15 20 25 30 35 40 45 2.5
0
vivo level
(µM)*
Cholesterol (µM)
40 40
2.0
10 2.0
in vivo level
in vivoinlevel
Urea (mM)*
Urea (mM)
20
Cholesterol
30 30
1.5 1.5
(days)
30
20 40
20
1.0 1.0
Doubling time
50
10 10
0.5
60 0.5
70
0 0 0
0 10 20 30 40 50 80 0 5 1010 15 20 20 25 30 30 35
40 40 45
50 0
Days 90
*µM/day/total cellular protein Days
Days *mM/da
100
in vivo
*Interestingly, urea production occurs via the alternate pathway because two genes (ornithine transcarbamylase and arginase I) of the ureaturnover 200-300
cycle have beendays
lost
from C3A cells. 60 60
5.0 5.0
50 50
12 4.5 4.5
l (µM)*
ol (µM)
4.0 40
4.0 40
level
level
level
3.5 3.5Figure 2. HEPG2/C3A spheroids seen in
phase contrast and dark field microscopy.
Not the low variability between the
spheroids. Note also, that light cannot
penetrate the spheroids, so it is necessary
to choose assays that are relevant for tissue
biopsies and not monolayer of cells.
Figure 3. Repeated APAP treatment of 21 days old HepG2/C3A spheroids with subsequent evaluation of ATP content at multiple concentrations. Black arrow
notes treatment point. (Fey, Korzeniowska and Wrzesinski, 2020).
Treatment of 21 day old HepG2/C3A spheroids with Acetaminophen
3.5
3
Acetaminophen**
2.5
0
2 0.625
ATP*
1.5 5
10
1
20
0.5
0
0 24 48 72 96 120 144 168 192 216 240 264
Time (h)
Time of treatment *Normalized to 1 and mg soluble cellular protein
**mg acetaminophen/mg soluble cellular protein
One of the roles of the liver is in the detoxification drug toxicity, eliminating the need for using animals for
of compounds. To investigate whether this can be this purpose (Fey, Korzeniowska and Wrzesinski, 2020).
modelled in vitro, 21 day old HEPG2/C3A spheroids were
treated with various does of acetaminophen (APAP, also
known as paracetamol) at two day intervals for 10 days
(black arrows). The effect on the cells was evaluate via
ATP content by the CellTiter-Glo assay from Promega.
Resources:
The highest dose (20mg APAP / mg soluble cellular
Wrzesinski et al. 2013
protein) killed the cells after one dose (red line –
Scan the QR Code
acute toxicity).
to see the article.
Halving this dose illustrated that multiple doses were
needed to kill the cells (blue line – chronic toxicity).
Fey, Korzeniowska
Halving the dose again illustrates that the HEPG2/
and Wrzesinski, 2020
C3A spheroids can respond to and recover from the
Scan the QR Code
treatment. Even at physiological doses (for example
to see the article.
for treating a headache), the HEPG2/C3A spheroids
still show a response and recover behaviour (0.625 mg/
mg, green line ). Similar response and recovery patterns
List of publications
have been seen for six drugs tested acetaminophen,
Scan the QR-code to access
amiodarone, diclofenac, metformin, phenformin and
a complete publication list.
valproic acid) and illustrate that HEPG2/C3A spheroids
can be used for the determination of repeated-dose
13BOOK YOUR
CLINOSTAR® DEMO
Are you ready to make the
transition to functional
3D cell culture?
Book a ClinoStar® demonstration now
and try cultivating spheroids or organoids in
your own lab with ClinoStar®.
Scan the QR code, fill out
your details and we will
contact you to arrange
a demonstration.
CONTACT US
If you have any questions about our products,
please ring our customer helpline
Telephone: (+45) 70 228 228
14PRODUCTDATA
ClinoStar® measurements
With door open 64 cm
FRONT
42 cm
SIDE
high
45 cm wide
FOOTPRINT
25 cm
depth
Weight: 23 kg
Internal diameter: 30.5 cms
Internal depth: 8 cms
ClinoStar® specifications
Door Safety
Open mechanism Push – click – swing open Paused while door is open UV-C emitting LED, fan and CO₂
Close mechanism Push to close - click Connectors
Axles USB Back and front (in door)
Capacity 6 axles Network Ethernet (RJ-45)
Speed range (rpm) 0 - 100 Ethernet port provide
C0 2
1500V insulation
Speed Accuracy ±1 %
Footprint
Direction Clockwise or anti clockwise
Control Independent Up to 3 units can be stacked on top
Space saving configuration of each other (the stacking bar must
Temperature data be used to increase their stability)
Temperature range From 6 to 20 ºC above ambient Electrical data
Temperature accuracy ± 0.25 ºC Rated Voltage [V] 100 - 240 V
CO 2 -data Power frequency [Hz] 50 - 60 Hz
CO₂ range [Vol.-% CO₂] 0 - 10 % Nominal power [A] 1.8 A
CO₂ measurement IR Power cord length 2 metres
CO₂ calibration Factory calibrated for 10 years Appliance Class Class I equipment
Monitoring Pollution Degree 2
Cameras 6 (placed opposite to each axle) Overvoltage category II
Camera resolution 5 Megapixel
Lighting Front and back LEDs for each axle
Decontamination
Incorporated method UV-C LED 300 mA ClinoReactor specifications
Time User activated (2 hours runtime) Catalogue #
Controller 10004-12
Device Tablet Size
Communication method Wi-Fi, Ethernet 12 x ClinoReactor with 10 mL culture chamber
Screen size 10,1" Description
Screen resolution 1920 x 1200
ClinoReactor is made from polypropolene and polystyrene. Supplied
Units to control 50 with 25 mL water for rehydration of water beads. 10 days use.
15© 2021 - Design: www.reklamehuset.dk CelVivo ApS Denmark Telephone: (+45) 70 228 228 info@celvivo.com
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